Programmed cell death (PCD) is a strictly regulated process and its disruption results in myriad developmental deficits and pathological sequelae. PCD is especially critical in the mammalian nervous system where its perturbation results in aberrant neural development and contributes to many neural disorders. There has been much research into the role of caspases in cell death. However, there is growing evidence for the importance of caspase-independent cell killing in the mammalian nervous system and other tissues. It is difficult to identify the components of the latter pathway or establish its contribution to cell elimination in vivo as it is intimately interwoven with, and masked by, the ubiquitous caspase cascades. We have developed a paradigm that can circumventthis limitation. CED-4S is a pro-apoptotic protein from C. elegans that is lethal when expressed in Saccharomyces cerevisiae. CED-4S lethality in yeast shows physiological specificity as it is blocked by its natural antagonist, CED-9 and is not mimicked by its anti-apoptotic splice variant, CED-4L. However, CED-4S toxicity in yeast does not require a caspase. Given the high degree of structural conservation amongst components of the cell suicide machinery, we propose to use CED-4S lethality in yeast as a paradigm to isolate molecules involved in caspase-independent killing. Subsequently, we will identify the mammalian counterparts of these molecules and investigate their function in the vertebrate nervous system. Using a CED-4S suppresser screen, we isolated 2 yeast AAA-ATPases, Cdc48 and yAPO-1 that have homologs in higher eukaryotes that have been implicated in neuronal death. Cdc48 binds to CED-4 whereas yAPO-l does not. This suggests a scenario in which CED-4S complexes with Cdc48 and alters its function, thereby leading to death. yAPO-l may have a redundant function with Cdc48 or it may lie downstream in the caspase-independent death pathway. Based upon these findings, we will use yeast and mammalian models to characterize the caspase-independent death pathway and determine the role that these and other CED-4 suppressers play in neuronal death in mice. In Specific Aim 1, we will determine the composition and functional domains of CED-4-containing complexes in yeast. In Specific Aim 2, downstream targets of CED-4 will be identified in yeast using a CED-4S suppresser screen. In Specific Aim 3, we will determine the expression and function of the mammalian homologs of the CED-4 suppressers in developing and adult brain and assess their contribution to normal and pathological cell death in the nervous system.